Mutant enterotoxin effective as a non-toxic oral adjuvant

ABSTRACT

Methods and compositions are provided herein for the se of a novel mutant form of  E. coli  heat-labile enterotoxin which has lost its toxicity but has retained its immunologic activity. This enterotoxin is used in combination with an unrelated antigen to achieve an increased immune response to said antigen when administered as part of an oral vaccine preparation.

This is a continuation, of application Ser. No. 08/296,848, filed Aug.26, 1994, which is now U.S. Pat. No. 6,019,982.

The research described in this specification was supported in part bythe United States Navy, Grant Number N00014-83-K-0192.

TABLE OF CONTENTS

1. FIELD OF THE INVENTION

2. BACKGROUND OF THE INVENTION

3. SUMMARY OF THE INVENTION

4. BRIEF DESCRIPTION OF THE FIGURES

5. DETAILED DESCRIPTION OF THE INVENTION

5.1 PRODUCTION OF mLT

5.2 MODE OF ADMINISTRATION OF mLT AND UNRELATED ANTIGEN

6. EXAMPLES

6.1 CONSTRUCTION OF mLT

6.2 EFFECT OF mLT ON Y-1 ADRENAL CELLS

6.3 ADP-RIBOSYLATING ENZYMATIC ACTIVITY OF mLT

6.4 ENTEROTOXIC ACTIVITY OF mLT 29

6.5 ADJUVANT ACTIVITY OF mLT

6.5.1 SERUM IgG ANTI-OVA

6.5.2 MUCOSAL sIgA ANTI-OVA

6.5.3 SERUM IgG ANTI-LT

6.5.4 MUCOSAL sIgA ANTI-LT

7. DEPOSIT OF MICROORGANISMS

1. FIELD OF THE INVENTION

The present invention is directed towards a genetically distinct mutantof E. coli heat-labile enterotoxin (LT) and its use as an oral adjuvantto induce mucosal and serum antibodies. Specifically, the mutant LT ismodified by a single amino acid substitution that abolishes its inherenttoxicity but leaves intact the adjuvant properties of the molecule.

2. BACKGROUND OF THE INVENTION

Microbial pathogens can infect a host by one of several mechanisms. Theymay enter through a break in the integument induced by trauma, they maybe introduced by vector transmission, or they may interact with amucosal surface. The majority of human pathogens initiate disease by thelast mechanism, i.e., following interaction with mucosal surfaces.Bacterial and viral pathogens that act through this mechanism first makecontact with the mucosal surface where they may attach and thencolonize, or be taken up by specialized absorptive cells (M cells) inthe epithelium that overlay Peyer's patches and other lymphoid follicles[Bockman and Cooper, 1973, Am. J. Anat. 136:455-477; Owen et al., 1986,J. Infect. Dis. 153:1108-1118]. Organisms that enter the lymphoidtissues-may be readily killed within the lymphoid follicles, therebyprovoking a potentially protective immunological response as antigensare delivered to immune cells within the follicles (e.g., Vibriocholerae). Alternatively, pathogenic organisms capable of survivinglocal defense mechanisms may spread from the follicles and subsequentlycause local or systemic disease (i.e., Salmonella spp., poliovirus,rotavirus in immunocompromised hosts).

Secretory IgA (sIgA) antibodies directed against specific virulencedeterminants of infecting organisms play an important role in overallmucosal immunity [Cebra et al., 1986, In: Vaccines 86, Brown et al.(ed.), Cold Spring Harbor Laboratory, New York. p.p. 129-133]. In manycases, it is possible to prevent the initial infection of mucosalsurfaces by stimulating production of mucosal sIgA levels directedagainst relevant virulence determinants of an infecting organism.Secretory IgA may prevent the initial interaction of the pathogen withthe mucosal surface by blocking attachment and/or colonization,neutralizing surface acting toxins, or preventing invasion of the hostcells. While extensive research has been conducted to determine the roleof cell mediated immunity and serum antibody in protection againstinfectious agents, less is known about the regulation, induction, andsecretion of sIgA. Parenterally administered inactivated whole-cell andwhole-virus preparations are effective at eliciting protective serum IgGand delayed type hypersensitivity reactions against organisms that havea significant serum phase in their pathogenesis (i.e., Salmonella typhi,Hepatitis B). However, parenteral vaccines are not effective ateliciting mucosal sIgA responses and are ineffective against bacteriathat interact with mucosal surfaces and do not invade (e.g., Vibriocholerae). There is, however, recent evidence that parenterallyadministered vaccines may be effective against at least one virus,rotavirus, that interacts primarily with mucosal surfaces [Conner etal., 1993, J. Virol. 67:6633-6641]. Protection is presumed to resultfrom transudation of antigen specific IgG onto mucosal surfaces forvirus neutralization. Therefore, mechanisms that stimulate both serumand mucosal antibodies are important for effective vaccines.

Oral immunization can be effective for induction of specific sIgAresponses if the antigens are presented to the T and B lymphocytes andaccessory cells contained within the Peyer's patches where preferentialIgA B-cell development is initiated. The Peyer's patches contain helperT (TH)-cells that mediate B-cell isotype switching directly from IgMcells to IgA B-cells. The patches also contain T-cells that initiateterminal B-cell differentiation. The primed B-cells then migrate to themesenteric lymph nodes and undergo differentiation, enter the thoracicduct, then the general circulation, and subsequently seed all of thesecretory tissues of the body, including the lamina propria of the gutand respiratory tract. IgA is then produced by the mature plasma cells,complexed with membrane-bound Secretory Component, and transported ontothe mucosal surface where it is available to interact with invadingpathogens [Strober and Jacobs, 1985, In: Advances in host defensemechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), RavenPress, New York. p.p. 1-30; Tomasi and Plaut, 1985, In: Advances in hostdefense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.),Raven Press, New York. p.p. 31-61]. The existence of this common mucosalimmune system explains in part the potential of live oral vaccines andoral immunization for protection against pathogenic organisms thatinitiate infection by first interacting with mucosal surfaces.

A number of strategies have been developed for oral immunization,including the use of attenuated mutants of bacteria (i.e., Salmonellaspp.) as carriers of heterologous antigens [Cárdenas and Clements, 1992,Clin. Microbiol. Rev. 5:328-342; Clements et al., 1992, In: RecombinantDNA Vaccines: Rationale and Strategy, Isaacson (ed.), Marcel Decker, NewYork. p.p. 293-321; Clements and Cárdenas, 1990, Res. Microbiol.141:981-993; Clements and El-Morshidy, 1984, Infect. Immun. 46:564-569],encapsulation of antigens into microspheres composed ofpoly-DL-lactide-glycolide (PGL), protein-like polymers-proteinoids[Sanitago et al., 1993, Pharmaceutical Research 10:1243-1247], gelatincapsules, different formulations of liposomes [Alving et al., 1986,Vaccine 4:166-172; Garcon and Six, 1993, J. Immunol. 146:3697-3702;Gould-Fogerite and Mannino, 1993, In: Liposome Technology 2nd Edition.Vol. III, Gregoriadis (ed.)], adsorption onto nanoparticles, use oflipophilic immune stimulating complexes (ISCOMS) [Mowat and Donachie,1991, Immunology Today 12:383-385], and addition of bacterial productswith known adjuvant properties [Clements et al., 1988, Vaccine6:269-277; Elson, 1989, Immunology Today 146:29-33; Lycke and Holmgren,1986, Immunology 59:301-308; Lycke et al., 1992, Eur. J. Immunol.22:2277-2281]. The two bacterial products with the greatest potential tofunction as oral adjuvants are cholera toxin (CT), produced by variousstrains of V. cholerae, and the heat-labile enterotoxin (LT) produced bysome enterotoxigenic strains of Escherichia coli. Although LT and CThave many features in common, these are clearly distinct molecules withbiochemical and immunologic differences which make them unique.

The extensive diarrhea of cholera is the result of a potentexo-enterotoxin which causes the activation of adenylate cyclase and asubsequent increase in intracellular levels of cyclic 3′-,5′-adenosinemonophosphate (cAMP). The cholera enterotoxin (CT) is an 84,000 daltonpolymeric protein composed of two major, non-covalently associated,immunologically distinct regions or domains (“cholera-A” and“cholera-B”) (Finkelstein and LoSpalluto, 1969, J. Exp. Med. 130:185-202). Of these, the 56,000 dalton region, or choleragenoid, isresponsible for binding of the toxin to the host cell membrane receptor,G_(M1) (galactosyl-N-acetylgalactosaminyl-(sialyl)-galactosyl-glucosylceramide), which is found on the surface of essentially all eukaryoticcells. Choleragenoid is composed of five non-covalently associatedsubunits, while the A region (27,000 daltons) is responsible for thediverse biological effects of the toxin.

The relationship of the two subunits of CT with respect to theimmunologic properties of the molecule has been a source of considerabledebate. On the one hand, CT is an excellent immunogen that provokes thedevelopment of both serum and mucosal antitoxin antibody responses whendelivered orally. This finding is not new in that cholera patients areknown to develop rises in titers of antitoxin antibodies duringconvalescence from clinical cholera (Finkelstein, 1975, Curr. Top.Microbiol. Immunol. 69: 137-196). One key finding of those investigatingthe nature of this response was the observation that CT, unlike mostother protein antigens, does not induce oral tolerance against itself(Elson and Ealding, 1984, J. Immunol. 133: 2892-2897; Elson and Ealding,1984, J. Immunol. 132: 2736-2741). This was also found to be true whenjust the B-subunit was fed to mice, an observation substantiated by thecholera vaccine field trials in Bangladesh in which oral immunizationwith B-subunit combined with killed whole cells gave rise to mucosal aswell as systemic antitoxin antibody responses (Svennerholm et al., 1984,J. Infect. Dis. 149: 884-893).

In addition to being a potent oral immunogen, CT has a number of otherreported immunologic properties. As indicated above, Elson and Ealding[Elson and Ealding, 1984, J. Immunol. 133: 2892-2897] observed thatorally administered CT does not induce tolerance against itself.Moreover, simultaneous oral administration of CT with a soluble proteinantigen, keyhole limpet hemocyanin (KLH), resulted in the development ofsecretory IgA responses against both CT and KLH and also abrogated theinduction of oral tolerance against KLH. These findings weresubsequently confirmed and extended by Lycke and Holmgren [Lycke andHolmgren, 1986, Immunology 59:301-308]. The confusion arises when oneattempts to define the role of the A and B subunits of CT with respectto the adjuvant properties of the molecule. The following observations,as summarized by Elson [Elson, 1989, Immunology Today 146:29-33], arethe basis for that confusion:

CT does not induce oral tolerance against itself [Elson and Ealding,1984, J. Immunol. 133: 2892-2897].

CT-B does not induce oral tolerance against itself [Elson and Ealding,1984, J. Immunol. 133: 2892-2897].

CT can prevent the induction of tolerance against other antigens withwhich it is simultaneously delivered and also serve as an adjuvant forthose antigens [Elson and Ealding, 1984, J. Immunol. 133: 2892-2897;Lycke and Holmgren, 1986, Immunology 59:301-308].

CT can act as and adjuvant for CT-B [Elson and Ealding, 1984, J.Immunol. 133: 2892-2897].

Heat aggregated CT has little toxicity but is a potent oral immunogen[Pierce et al., 1983, Infect. Immun. 40: 1112-1118].

CT-B can serve as an immunologic “carrier” in a traditionalhapten-carrier configuration [Cebra et al., 1986, In: Vaccines 86, Brownet al. (ed.), Cold Spring Harbor Laboratory, New York. p.p. 129-133;McKenzie and Halsey, 1984, J. Immunol. 133: 1818-1824].

A number of researchers have concluded from these findings that theB-subunit must possess some inherent adjuvant activity. The findings ofCebra et al. [Cebra et al., 1986, In: Vaccines 86, Brown et al. (ed.),Cold Spring Harbor Laboratory, New York. p.p. 129-133], Lycke andHolmgren [Lycke and Holmgren, 1986, Immunology 59:301-308], and Liang etal. [Liang et al., 1988, J. Immunol. 141: 1495-1501] would argue againstthat conclusion. Cebra et al. [Cebra et al., 1986, In: Vaccines 86,Brown et al. (ed.), Cold Spring Harbor Laboratory, New York. p.p.129-133] demonstrated that purified CT-B was effective at raising thefrequency of specific anti-cholera toxin B-cells in Peyer's patches whengiven intraduodenally but, in contrast to CT, did not result insignificant numbers of IgA committed B-cells. Lycke and Holmgren [Lyckeand Holmgren, 1986, Immunology 59:301-308] compared CT and CT-B for theability to enhance the gut mucosal immune response to KLH by measuringimmunoglobulin secreting cells in the lamina propria of orally immunizedmice. They found no increase in anti-KLH producing cells in response toany dose of B-subunit tested in their system. Finally, Liang et al.[Liang et al., 1988, J. Immunol. 141: 1495-1501] found no adjuvanteffect when CT-B was administered orally in conjunction with inactivatedSendai virus.

Where adjuvant activity has been observed for isolated B-subunit, it hastypically been for one of two reasons. First, a traditional method ofpreparing B-subunit has been to subject holotoxin to dissociationchromatography by gel filtration in the presence of a dissociating agent(i.e., guanidine HCl or formic acid). The isolated subunits are thenpooled and the dissociating agent removed. B-subunit prepared by thistechnique is invariably contaminated with trace amounts of A-subunitsuch that upon renaturation.a small amount of holotoxin isreconstituted. The second reason has to do with the definition of animmunologic carrier. Like many other soluble proteins, B-subunit canserve as an immunologic vehicle for presentation of antigens to theimmune system. If those antigens are sufficiently small as to be poorlyimmunogenic, they can be made immunogenic in a traditionalhapten-carrier configuration. Likewise, there is a “theoretical” immuneenhancement associated with B-subunit, especially for oral presentation,in that B-subunit binds to the surface of epithelial cells and mayimmobilize an attached antigen for processing by the gut associatedlymphoid tissues. However, any potential advantage to this mechanism ofantigen stabilization may be offset by the distribution of the antigenacross non-immunologically relevant tissues, i.e., the surface ofintestinal epithelial cells. In context of the mucosal responsiveness,the immunologically relevant sites are the Peyer's patches, especiallyfor antigen-specific T cell-dependent B cell activation [Strober andJacobs, 1985, In: Advances in host defense mechanisms. Vol. 4. MucosalImmunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 1-30;Tomasi and Plaut, 1985, In: Advances in host defense mechanisms. Vol. 4.Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p.31-61; Brandtzaeg, 1989, Curr. Top. Microbiol. Immunol. 146: 13-25].Thus, the events up to isotype switching from IgM cells to IgA B-cellsoccurs in the Peyer's patches. Antigens localized on the epithelial cellsurface may contribute to antigen induced B cell proliferation in thatthe class II positive villous epithelial cells may act as antigenpresenting cells for T cell activation at the secretory site, therebyincreasing cytokihe production, terminal B cell differentiation,increased expression of secretory component, and increased externaltransport of antigen specific IgA [Tomasi, T. B., and A. G. Plaut. 1985,In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity,Gallin and Fauci (ed.), Raven Press, New York. p.p. 31-61]. Therelationships of these events have not been clearly defined forB-subunit as a carrier of other antigens and use of the term “adjuvant”would seem inappropriate for such an effect.

It is clear that the adjuvant property of the molecule resides in theholotoxin in which B-subunit is required for receptor recognition and tofacilitate penetration of the A-subunit into the cell. The A-subunit isalso required for adjuvant activity, presumably as a function of itsADP-ribosylating enzymatic activity and ability to increaseintracellular levels of cAMP (see below). The B-subunit alone may act asa carrier of other antigens in that when conjugated to those antigensthey can be immobilized for processing by the gut associated lymphoidtissues.

Although LT and CT have many features in common, these are clearlydistinct molecules with biochemical and immunologic differences whichmake them unique, including a 20% difference in nucleotide and aminoacid sequence homology [Dallas and Falkow, 1980, Nature 288: 499-501].The two toxins have the same subunit number and arrangement, samebiological mechanism of action, and the same specific activity in manyin vitro assays [Clements and Finkelstein, 1979, Infect. Immun.24:760-769; Clements et al., 1980, Infect. Immun. 24: 91-97].

There are, however, significant differences between these molecules thatinfluence not only their enterotoxic properties, but also their abilityto function as adjuvants. To begin with, unlike CT produced by V.cholerae, LT remains cell associated and is only released from E. coliduring cell lysis [Clements and Finkelstein, 1979, Infect. Immun.24:760-769]. CT is secreted from the vibrio as soon as it is synthesizedand can be readily identified in, and purified from, culturesupernatants. Consequently, in contrast to CT, LT is not fullybiologically active when first isolated from the cell. Consistent withthe A-B model for bacterial toxins, LT requires proteolysis anddisulfide reduction to be fully active. In-the absence of proteolyticprocessing, the enzymatically active A₁ moiety is unable to dissociatefrom the A₂ component and cannot reach its target substrate (adenylatecyclase) on the basolateral surface of the intestinal epithelial cell.This is also true for CT, but proteases in the culture supernatant, towhich the toxin is exposed during purification, perform the proteolysis.Since LT is not fully biologically active, it is difficult to identifyduring purification using in vitro biological assays such as the Y-1adrenal cell assay or permeability factor assay.

This difference in activation of the isolated material results indifferences in response thresholds for LT and CT in biologic systems.For instance, CT induces detectable net fluid secretion in the mouseintestine at a dose of 5-10 μg. LT induces detectable net secretion inthe mouse intestine at levels above 100 μg. In the rabbit ligated ilealloop, the difference is dramatic and clear cut. Moreover, in primates LThas been shown not to induce fluid secretion at any dose tested up to 1milligram. This is 200 times the amount of CT reported to inducepositive fluid movement in humans. When LT is exposed to proteolyticenzymes with trypsin-like specificity, the molecule becomesindistinguishable from CT in any biologic assay system. This wasdemonstrated clearly by Clements and Finkelstein [Clements andFinkelstein, 1979, Infect. Immun. 24:760-769].

In addition to the above reported differences, LT has an unusualaffinity for carbohydrate containing matrices. Specifically, LT, with amolecular weight of 90,000, elutes from Sephadex columns (glucose) withan apparent molecular weight of 45,000 and from Agarose columns(galactose) with an apparent molecular weight of 0. That is, it binds togalactose containing matrices and can be eluted from those matrices inpure form by application of galactose. LT binds not only to agarose incolumns used for purification, but more importantly, to other biologicalmolecules containing galactose, including glycoproteins andlipopolysaccharides.

This lectin-like binding property of LT results in a broader receptordistribution on mammalian cells for LT than for CT which binds only toG_(M1). This may account in part for the reported differences in theabilities of these two molecules to induce different helper T lymphocyteresponses [McGhee et al., 1994, Mucosal Immunology Update, Spring 1994,Raven Press, New York. p. 21].

In these studies reported by McGhee et al. (McGhee et al., 1994, MucosalImmunology Update, Spring 1994, Raven Press, New York. p. 21), it wasshown that oral immunization of mice with vaccines such as tetanustoxoid (TT) with CT as a mucosal adjuvant selectively induces T_(H)2type cells in Peyer's patches and spleens as manifested by TH cellswhich produce IL-4 and IL-5, but not IL-2 or INF-gamma. (For a morecomplete review of the cytokine network see Arai et al., 1990, Ann. Rev.Biochem. 59:783-836). Importantly, when CT was used as a mucosaladjuvant it also enhanced antigen-specific IgE responses in addition tothe IgA response. Such enhancement of IgE responses seriouslycompromises the safety of CT as a mucosal adjuvant due to the prospectof inducing immediate-type hypersensitivity reactions. In contrast, LTinduces both T_(H)1 and T_(H)2 cells and predominantly antigen-specificIgA responses without IgE responses when used as an orally administeredmucosal adjuvant.

The two molecules also have many immunologic differences, asdemonstrated by immunodiffusion studies [Clements and Finkelstein, 1978,Infect. Immun. 21: 1036-1039; Clements and Finkelstein, 1978, Infect.Immun. 22: 709-713], in vitro neutralization studies, and the partialprotection against LT associated E. coli diarrhea in volunteersreceiving B-subunit whole cell cholera vaccine [Clemens et al., 1988, J.Infect. Dis. 158: 372-377].

Taken together, these findings demonstrate that LT and CT are uniquemolecules, despite their apparent similarities, and that LT is apractical oral adjuvant while CT is not. grew out of an investigation ofthe influence of LT on the development of tolerance to orallyadministered antigens by one of the present inventors. It was not clearwhether or not LT would also influence the induction of oral toleranceor exhibit the adjuvant effects demonstrated for CT, given theobserved.differences between the two molecules. Consequently, thepresent inventors examined a number of parameters, including the effectof LT on oral tolerance to OVA and the role of the two subunits of LT inthe observed response, the effect of varying the timing and route ofdelivery of LT, the effect of prior exposure to OVA on the ability of LTto influence tolerance to OVA, the use of LT as an adjuvant with twounrelated antigens, and the effect of route of immunization on anti-OVAresponses. The results obtained from these studies [Clements et al.,1988, Vaccine 6:269-277; Clements et al., 1988, Abstract No. B91, 88thAnn. Meet. Am. Soc. Microbiol.] are summarized below:

1.Simultaneous administration of LT with OVA was shown to prevent theinduction of tolerance to OVA and to increase the serum anti-OVA IgGresponse 30 to 90 fold over OVA primed and PBS. primed animals,respectively. This effect was determined to be a function of theenzymatically active A-subunit of the toxin since the B-subunit alonewas unable to influence tolerance induction.

2.Animals fed LT with OVA after an initial OVA prime developed asignificantly lower serum IgG and mucosal IgA anti-OVA response. thanthose fed LT with OVA in the initial immunization, indicating that priorexposure to the antigen reduces the effectiveness of LT to influencetolerance and its ability to act as an adjuvant. LT was not able toabrogate tolerance once it had been established. This was also found tobe true for CT when animals were pre-immunized with OVA prior to oralovalbumin plus CT and offers some insight into the beneficialobservation that antibody responses to nontarget dietary antigens arenot increased when these adjuvants are used.

3.Serum IgG and mucosal IgA responses in animals receiving LT on only asingle occasion, that being upon first exposure to antigen, wereequivalent to responses after three OVA/LT primes, indicating thatcommitment to responsiveness occurs early and upon first exposure toantigen. It was also demonstrated that the direction of the response toeither predominantly serum IgG or mucosal IgA can be controlled bywhether or not a parenteral booster dose is administered.

4.Simultaneous administration of LT with two soluble protein antigensresults in development of serum and mucosal antibodies against bothantigens if the animal has no prior immunologic experience with either.This was an important finding since one possible application of LT as anadjuvant would be for the development of mucosal antibodies againstcomplex antigens, such as killed bacteria or viruses, where the abilityto respond to multiple antigens would be important.

Studies by Tamura.et al., [Tamura et al., U.S. Pat. No. 5,182,109]demonstrated that LT and/or CT administered intranasally enhanced theantibody titer against a co-administered antigen. However, nowhere inTamura et al. is it taught that these toxins can induce a protectiveimmune response when administered orally.

Clearly, LT has significant immunoregulatory potential, both as a meansof preventing the induction of tolerance to specific antigens and as anadjuvant for orally administered antigens and it elicits the productionof both serum IgG and mucosal IgA against antigens with which it isdelivered. This raises the possibility of an effective immunizationprogram against a variety of pathogens involving the oral administrationof killed or attenuated agents or relevant virulence determinants ofspecific agents. However, the fact that this “toxin” can stimulate a netlumenal secretory response when proteolytically cleaved, as by gutproteases, or when administered in high enough concentrations orally,may hinder investigation into its potential or prevent its use underappropriate conditions. This problem could be resolved if LT could be“detoxified” without diminishing the adjuvant properties of themolecules. It order to appreciate how this might be accomplished, it isnecessary to further analyze the mechanism of action of the LT and CTand the structural and functional relationships of these molecules. Asindicated previously, both LT and CT are synthesized as multisubunittoxins with A and B components. After the initial interaction of thetoxin with the host cell membrane receptor, the B region facilitates thepenetration of the A-subunit through the cell membrane. On thiolreduction, this A component dissociates into two smaller polypeptidechains. One of these, the A₁ piece, catalyzes the ADP-ribosylation ofthe stimulatory GTP-binding protein (G_(S)) in the adenylate cyclaseenzyme complex on the basolateral surface of the epithelial cell andthis results in increasing intracellular levels of cAMP. The resultingincrease in cAMP causes secretion of water and electrolytes into thesmall intestine through interaction with two cAMP-sensitive iontransport mechanisms involving 1) NaCl co-transport across the brushborder of villous epithelial cells, and 2) electrogenic Na⁺ dependentCl⁻ secretion by crypt cells [Field, 1980, In: Secretory diarrhea, Fieldet al. (ed.), Waverly Press, Baltimore. p.21-30]. The A subunit is alsothe principal moiety associated with immune enhancement by these toxins.This subunit then becomes a likely target for manipulation in order todissociate the toxic and immunologic functions of the molecules. Arecent report by Lycke et al. [Lycke et al., 1992, Eur. J. Immunol.22:2277-2281] makes it clear that alterations that affect theADP-ribosylating enzymatic activity of the toxin and alter the abilityto increase intracellular levels of cAMP also prevent the molecule fromfunctioning as an adjuvant. Consequently, another approach todetoxification must be explored.

3. SUMMARY OF THE INVENTION

The present invention is based on the surprising observation that amutant form of LT, which has lost its toxic effect and is devoid ofADP-ribosyltransferase activity, still retains its activity as animmunological adjuvant. The mutant form of LT differs from the wild-typeby a single amino acid substitution, Arg₁₉₂-Gly₁₉₂, rendering a trypsinsensitive site insensitive. The loss of the proteolytic site preventsthe proteolytic processing of the A subunit into its toxic form. NativeLT is not toxic when first isolated from the bacterium but has thepotential to be fully toxic when exposed to proteases such as thosefound in the mammalian intestine. The mutant form of LT no longer hasthe potential to become toxic due to proteolytic activation. This mutantLT (hereinafter mLT) retains the capability of enhancing an animal'simmune response (e.g., IgG, IgA) to an antigen unrelated to LT or mLTwith no toxic side effects. Experimental evidence shows that mLT hasutility as an adjuvant for orally administered antigens; suchadministration results in the production of serum IgG and/or mucosalsIgA against the antigen with which the mLT is delivered. The presentinvention provides a method for induction of a serum and/or mucosalimmune response in a host to any orally administered antigen whichcomprises administering to the host an effective amount of mLT inconjunction with oral administration of an effective amount of theantigen. Preferably, the antigen and the mLT are administered initiallyin a simultaneous dose.

The present method and compositions provide an improved mode of oralimmunization for development of serum and mucosal antibodies againstpathogenic microorganisms. Production of IgA antibody responses againstpathogenic microorganisms which penetrate or invade across mucosalsurfaces can be directed to that surface, while a significant serumantibody response can be developed to prevent infection by pathogenicmicroorganisms against which serum antibody is protective. The presentinvention is useful for any specific antigen where a specificneutralizing antibody response would be useful in ablating thephysiological or disease state associated with that antigen.

The present invention also provides a composition useful as a componentof a vaccine against enterotoxic bacterial organisms expressingcholera-like enterotoxins and methods for its use.

The invention also provides a composition useful in these methods. Thecomposition comprises an effective amount of mLT in combination with aneffective amount of antigen.

4. BRIEF DESCRIPTION OF THE FIGURES

The present invention may be understood more fully by reference to thefollowing detailed description of the invention, examples of specificembodiments of the invention and the appended figures in which:

FIG. 1. Schematic diagram of-the plasmid pBD94, which encodes bothsubunits A and B under the control of the lac promoter. Plasmid pBD95contains the single base substitution at amino acid residue 192 ofsubunit A, coding for Gly rather than Arg, which preserves the readingframe but eliminates the proteolytic site. The amino acid sequencecorresponding to the region of trypsin sensitivity and the site of theamino acid substitution Arg₁₉₂-Gly₁₉₂ is shown.

FIG. 2. Graphic demonstration of the dose-dependent increase in thelevels of ADP-ribosylagmatine as a function of increasing amounts of CT.

FIG. 3. Fluid accumulation after feeding 125 μg of native LT but notafter feeding 125 μg of mLT to mice. The gut-carcass ratio is defined asthe intestinal weight divided by the remaining carcass weight.

FIG. 4. Ability of mLT to act as an immunological adjuvant. FIG. 4A,Ability of mLT to induce a serum IgG response to OVA. FIG. 4B, Abilityof mLT to induce a mucosal sIgA response to OVA.

FIG. 5. Experimental demonstration that mLT retains the ability toprevent induction of oral tolerance to LT. FIG. 5A, Ability of mLT toinduce a serum IgG response to LT. FIG. 5B, Ability of mLT to induce amucosal sIgA response to LT.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses a composition and methods for its useto promote the production of mucosal and serum antibodies againstantigens that are simultaneously orally administered with a geneticallymodified bacterial toxin. The modified toxin is a form of theheat-labile enterotoxin (LT) of E. coli which through geneticengineering has lost its trypsin sensitive site rendering the moleculenon-toxic but yet, unexpectedly, retains its ability to act as animmunological adjuvant. The mutant LT is herein termed “mLT”. Theinvention is based on the discovery that mLT is as effective as LT as animmunological adjuvant, an unexpected and surprising result. mLT nolonger has the enzymatic activity of ADP-ribosylation because the Asubunit can no longer be proteolytically processed. In contrast topublished studies of Lycke and colleagues, which made it clear thatalterations that effect the ADP-ribosylating activity of LT also preventthe molecule from functioning as an immunologic adjuvant [Lycke et al.,1992, Eur. J. Immunol. 22:2277-2281], the presently described mLTretains activity as an immunological adjuvant although, as demonstratedin the examples, it does not have ADP-ribosylating activity.

The novel mutant form of the heat-labile enterotoxin of E. coli, mLT,described herein, behaves as an adjuvant and elicits the production ofboth serum IgG and mucosal sIgA against antigens with which it isdelivered. The utility of this surprising discovery is that an adjuvanteffective amount of mLT may be utilized in an effective immunizationprogram against a variety of pathogens involving the oral administrationof an effective amount of mLT adjuvant in admixture with killed orattenuated pathogens or relevant virulence determinants of specificpathogens with no fear of the real or potential toxic side-effectsassociated with oral administration of CT or LT.

The present invention supersedes the prior art in that the presentinvention may be used in a variety of immunological applications whereCT, LT, or subunits of CT or LT may have been used, but now with mLTthere are no real or potential side-effects, such as diarrhea,associated with its use. In contrast to LT, which although not toxicwhen first isolated from the bacterium, has the potential to be fullytoxic when exposed to proteases such as those found in the mammalianintestine, mLT does not have the potential to become toxic due toproteolytic activation.

Another embodiment of the present invention is as a component of avaccine against enterotoxic organisms which express cholera-like toxins.The present inventors have shown that mLT is not subject to orallyinduced immune tolerance when administered (see below), therefore mLTcan function and is highly desired as a component of vaccines directedagainst enterotoxic organisms. Current technology provides for vaccinesagainst cholera-like toxin expressing organisms containing killed wholecells and the B subunit of the toxin. By replacing the B subunit withmLT in the vaccine, the vaccine is improved in two different ways.First, mLT, which has both the A and B subunits will now induce animmune esponse not only to the B subunit but to the A subunit as well.This provides for more epitopes for effective neutralization. Second,the adjuvant activity inherent in mLT will enhance the immune responseagainst the killed whole cell component of the vaccine.

Further, other investigators [Häse et al., 1994, Infect. Immun.62:3051-3057] have shown that the A subunit, modified so that it is nolonger toxic by altering the active site of the ADP-ribosylatingenzymatic activity, (as opposed to the proteolytic site which is thesubject of the current invention) can induce an immune response againstthe wild type A subunit. However, the A subunit so modified now lacksimmunologic adjuvant activity and is therefore less desirable as avaccine component than mLT.

Moreover, since antibodies against mLT cross-react with LT and CT, mLTcan be used in vaccines directed against many types of enterotoxicbacterial organisms that express cholera-like toxins, such asEscherichia spp. and Vibrio spp.

5.1 Production of mLT

The wild-type LT toxin is encoded on a naturally occurring plasmid foundin strains of enterotoxigenic E. coli capable of producing this toxin.The present inventors had previously cloned the LT gene from a humanisolate of E. coli designated H10407. This subclone consists of a 5.2 kbDNA fragment from the enterotoxin plasmid of H10407 inserted into thePstI site of plasmid pBR322 [Clements et al, 1983, Infect. Immun.40:653]. This recombinant plasmid, designated pDF82, has beenextensively characterized and expresses LT under control of the nativeLT promoter. The next step in this process was to place the LT geneunder the control of a strong promoter, in this case the lac promoter onplasmid pUC18. This was accomplished by isolating the genes for LT-A andLT-B separately and recombining them in a cassette in the vectorplasmid. This was an important step because it permitted purification ofreasonable quantities of LT and derived mutants for subsequent analysis.This plasmid, designated pBD94, is shown diagrammatically in FIG. 1.

Both CT and LT are synthesized with a trypsin sensitive peptide bondthat joins the A₁ and A2 pieces. This peptide bond must be nicked forthe molecule to be “toxic”. This is also true for diphtheria toxin, theprototypic A-B toxin, and for a variety of other bacterial toxins. Ifthe A₁—A₂ bond is not removed, either by bacterial proteases orintestinal proteases in the lumen of the bowel, the A₁ piece cannotreach its target on the basolateral surface of the intestinal epithelialcell. In contrast to CT, LT is not fully biologically active when firstisolated from the cell. LT also requires proteolysis to be fully activeand the proteolytic activation does not occur inside of the bacterium.Therefore, one means of altering the toxicity of the molecule withoutaffecting the ADP-ribosylating enzymatic activity would be to remove bygenetic manipulation the trypsin sensitive amino acids that join the A₁and A₂ components of the A subunit. If the molecule cannot beproteolytically cleaved, it will not be toxic. One skilled in theart.would predict that the molecule should, however, retain itsADP-ribosylating enzymatic activity and consequently, its adjuvantfunction.

FIG. 1 shows the sequence of the disulfide subtended region thatseparates the A₁ and A₂ pieces. Within this region is a single Arginineresidue which is believed to be the site of cleavage necessary toactivate the toxic properties of the molecule. This region was changedby site-directed mutagenesis in such a way as to render the moleculeinsensitive to proteolytic digestion and, consequently, nontoxic.

Site-directed mutagenesis is accomplished by hybridizing to singlestranded DNA a synthetic oligonucleotide which is complementary to thesingle stranded template except for a region of mismatch near thencenter. It is this region that contains the desired nucleotide change orchanges. Following hybridization with the single stranded target DNA,the oligonucleotide is extended with DNA polymerase to create a doublestranded structure. The nick is then sealed with DNA ligase and theduplex structure is transformed into an E. coli host. The theoreticalyield of mutants using this procedure is 50% due to thesemi-conservative mode of DNA replication. In practice, the yield ismuch lower. There are, however, a number of methods available to improveyield and to select for oligonucleotide directed mutants. The systememployed utilized a second mutagenic oligonucleotide to create alteredrestriction sites in a double mutation strategy.

The next step was to substitute another amino acid for Arg (i.e.,GGA=Gly replaces AGA=Arg), thus preserving the reading frame whileeliminating the proteolytic site. mLT was then purified by agaroseaffinity chromatography from one mutant (pBD95) which had been confirmedby sequencing. Alternate methods of purification will be apparent tothose skilled in the art. This mutant LT, designated LT(_(R192G)) wasthen examined by SDS-polyacrylamide gel electrophoresis for modificationof the trypsin sensitive bond. Samples were examined with and withoutexposure to trypsin and compared with native (unmodified) LT. mLT doesnot dissociate into A₁ and A₂ when incubated with trypsin, therebyindicating that sensitivity to protease has been removed.

5.2 Mode of Administration of mLT and Unrelated Antigens

In accordance with the present invention, mLT can be administered inconjunction with any biologically relevant antigen and/or vaccine, suchthat an increased immune response to said antigen and/or vaccine isachieved. In a preferred embodiment, the mLT and antigen areadministered simultaneously in a pharmaceutical composition comprisingan effective amount of mLT and an effective amount of antigen. The modeof administration is oral. The respective amounts of mLT and antigenwill vary depending upon the identity of the antigen employed and thespecies of animal to be immunized. In one embodiment, the initialadministration of mLT and antigen is followed by a boost of the relevantantigen. In another embodiment no boost is given. The timing of boostingmay vary, depending on the antigen and the species being treated. Themodifications in dosage range and timing of boosting for any givenspecies and antigen are readily determinable by routine experimentation.The boost may be of antigen alone or in combination with mLT. The modeof administration of the boost may either be oral, nasal, or parenteral;however, if mLT is used in the boost, the administration is preferablyoral.

The methods and compositions of the present invention are intended foruse both in immature and mature vertebrates, in particular birds,mammals, and humans. Useful antigens, as examples and not by way oflimitation, would include antigens from pathogenic strains of bacteria(Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae,Neisseria meningitidis, Corynebacterium diphtheriae, Clostridiumbotulinum, Clostridium perfringens, Clostridium tetani, Hemophilusinfluenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiellarhinoscleromotis, Staphylococcus aureus, Vibrio colerae, Escherichiacoli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonashydrophila, Bacillus cereus, Edwardsiella tarda, Yersiniaenterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigelladysenteriae, Shigellaflexneri, Shigella sonnei, Salmonella typhimurium,Treponema pallidum, Treponema pertenue, Treponema carateneum, Borreliavincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii,Francisella tularensis, Brucella abortus, Brucella suis, Brucellamelitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis,Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis,Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoebahistolytica, Trichomonas tenas, Trichomonas hominis, Trichomonasvaginalis, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosomacruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis,Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum,Plasmodium malaria); or Helminiths (Enterobius vermicularis, Trichuristrichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloidesstercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosomahaematobium, and hookworms) either presented to the immune system inwhole cell form or in part isolated from media cultures designed to growsaid organisms which are well known in the art, or protective antigensfrom said organisms obtained by genetic engineering techniques or bychemical synthesis.

Other relevant antigens would be pathogenic viruses (as examples and notby limitation: Poxviridae, Herpesviridae, Herpes Simplex virus 1, HerpesSimplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae,Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenzaviruses, parainfluenza viruses, mumps, measles, respiratory syncytialvirus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis Avirus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus,Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae,and Human Immunodeficiency Virus) either presented to the immune systemin whole or in part isolated from media cultures designed to grow suchviruses which are well known in the art: or protective antigenstherefrom obtained by genetic engineering techniques or by chemicalsynthesis.

Further examples of relevant antigens include, but are not limited to,vaccines. Examples of such vaccines include, but are not limited to,influenza vaccine, pertussis vaccine, diphtheria and tetanus toxoidcombined with pertussis vaccine, hepatitis A vaccine, hepatitis Bvaccine, hepatitis C vaccine, hepatitis E vaccine, Japanese encephalitisvaccine, herpes vaccine, measles vaccine, rubella vaccine, mumpsvaccine, mixed vaccine of measles, mumps and rubella, papillomavirusvaccine, parvovirus vaccine, respiratory syncytial virus vaccine, Lymedisease vaccine, polio vaccine, malaria vaccine, varicella vaccine,gonorrhea vaccine, HIV vaccine, schistosomiasis vaccine, rota vaccine,mycoplasma vaccine pneumococcal vaccine, meningococcal vaccine andothers. These can be produced by known common processes. In general,such vaccines comprise either the entire organism or virus grown andisolated by techniques well known to the skilled artisan or compriserelevant antigens of these organisms or viruses which are produced bygenetic engineering techniques or chemical synthesis. Their productionis illustrated by, but not limited to, as follows:

Influenza vaccine: a vaccine comprising the whole or part ofhemagglutinin, neuraminidase, nucleoprotein and matrix protein which areobtainable by purifying a virus, which is grown in embryonated eggs,with ether and detergent, or by genetic engineering techniques orchemical synthesis.

Pertussis vaccine: a vaccine comprising the whole or a part of pertussistoxin, hemagglutinin and K-agglutin which are obtained from avirulenttoxin with formalin which is extracted by salting-out orultracentrifugation from the culture broth or bacterial cells ofBordetella pertussis, or by genetic engineering techniques or chemicalsynthesis.

Diphtheria and tetanus toxoid combined with pertussis vaccine: a vaccinemixed with pertussis vaccine, diphtheria and tetanus toxoid.

Japanese encephalitis vaccine: a vaccine comprising the whole or part ofan antigenic protein which is obtained by culturing a virusintracerebrally in mice and purifying the virus particles bycentrifugation or ethyl alcohol and inactivating the same, or by geneticengineering techniques or chemical synthesis.

Hepatitis B vaccine: a vaccine comprising the whole or part of anantigen protein which is obtained by isolating and purifying the HBsantigen by salting-out or ultracentrifugation, obtained from hepatitiscarrying blood, or by genetic engineering techniques or by chemicalsynthesis.

Measles vaccine: a vaccine comprising the whole or part of a virus grownin a cultured chick embryo cells or embryonated egg, or a protectiveantigen obtained by genetic engineering or chemical synthesis.

Rubella vaccine: a vaccine comprising the whole or part of a virus grownin cultured chick embryo cells or embryonated egg, or a protectiveantigen obtained by genetic engineering techniques or chemicalsynthesis.

Mumps vaccine: a vaccine comprising the whole or part of a virus grownin cultured rabbit cells or embryonated egg, or a protective antigenobtained by genetic engineering techniques or chemical synthesis.

Mixed vaccine of measles, rubella and mumps: a vaccine produced bymixing measles, rubella and mumps vaccines.

Rota vaccine: a vaccine comprising the whole or part of a virus grown incultured MA 104 cells or isolated from the patient's feces, or aprotective antigen obtained by genetic engineering techniques orchemical synthesis.

Mycoplasma vaccine: a vaccine comprising the whole or part of mycoplasmacells grown in a liquid culture medium for mycoplasma or a protectiveantigen obtained by genetic engineering techniques.or chemicalsynthesis.

Those conditions for which effective prevention may be achieved by thepresent method will be obvious to the skilled artisan.

The vaccine preparation compositions of the present invention can beprepared by mixing the above illustrated antigens and/or vaccines withmLT at a desired ratio. The preparation should be conducted strictlyaseptically, and each component should also be aseptic. Pyrogens orallergens should naturally be removed as completely as possible. Theantigen preparation of the present invention can be used by preparingthe antigen per se and the mLT separately.

Further, the present invention encompasses a kit comprising an effectiveamount of antigen and an adjuvant effective amount of mLT. In use, thecomponents of the kit can either first be mixed together and thenadministered orally or the components can be administered orallyseparately within a short time of each other.

The vaccine preparation compositions of the present invention can becombined with either a liquid or solid pharmaceutical carrier, and thecompositions can be in the form of tablets, capsules, powders, granules,suspensions or solutions. The compositions can also contain suitablepreservatives, coloring and flavoring agents, or agents that produceslow release. Potential carriers that can be used in the preparation ofthe pharmaceutical compositions of this invention include, but are notlimited to, gelatin capsules, sugars, cellulose derivations such assodium carboxymethyl cellulose, gelatin, talc, magnesium stearate,vegetable oil such as peanut oil, etc., glycerin, sorbitol, agar andwater. Carriers may also serve as a binder to facilitate tabletting ofthe compositions for convenient oral administration.

The vaccine preparation composition of this invention may be maintainedin a stable storage form for ready use by lyophilization or by othermeans well known to those skilled in the art. For oral administration,the vaccine preparation may be reconstituted as a suspension in bufferedsaline, milk, or any other physiologically compatible liquid medium. Themedium may be made more palatable by the addition of suitable coloringand flavoring agents as desired.

Administration of the vaccine preparation compositions may be precededby an oral dosage of an effective amount of a gastric acid neutralizingagent. While many compounds could be used for this purpose, sodiumbicarbonate is preferred. Alternatively, the vaccine compositions may bedelivered in enteric coated capsules (i.e., capsules that dissolve onlyafter passing through the stomach).

6. EXAMPLES

The following examples are presented for purposes of illustration onlyand are not intended to limit the scope of the invention in any way.

6.1 Construction of mLT

The wild-type LT toxin is encoded on a naturally occurring plasmid foundin strains of enterotoxigenic E. coli capable of producing this toxin.The present inventors had previously cloned the LT gene from a humanisolate of E. coli designated H10407. This subelone consists of a 5.2 kbDNA fragment from the enterotoxin plasmid of H10407 inserted into thePstI site of plasmid pBR322 [Clements et al., 1983, Infect. Immun.40:653]. This recombinant plasmid, designated pDF82, has beenextensively characterized and expresses LT under control of the nativeLT-promoter. The next step in this process was to place the LT geneunder the control of a strong promoter, in this case the lac promoter onplasmid pUC18. This was accomplished by isolating the genes for LT-A andLT-B separately and recombining them in a cassette in the vectorplasmid. This was an important step because it permitted purification ofreasonable quantities of LT and derived mutants for subsequent analysis.This plasmid, designated pDF94, is shown diagrammatically in FIG. 1.

Both CT and LT are synthesized with a trypsin sensitive peptide bondthat joins the A₁ and A₂ pieces. This peptide bond must be nicked forthe molecule to be “toxic”. This is also true for diphtheria toxin, theprototypic A-B toxin, and for a variety of other bacterial toxins. Ifthe A₁—A₂ bond is not removed, either by bacterial proteases orintestinal proteases in the lumen of the bowel, i.e., proteolyticprocessing or activation, the A₁ piece cannot reach its target on thebasolateral surface of the intestinal epithelial cell. In contrast toCT, LT is not fully biologically active when first isolated from thecell. LT also requires proteolysis to be fully active and theproteolytic activation does not occur inside of the bacterium.Therefore, one means of altering the toxicity of the molecule withoutaffecting the ADP-ribosylating enzymatic activity would be to remove bygenetic manipulation the trypsin sensitive amino acids that join the A₁and A₂ components of the A subunit. If the molecule cannot beproteolytically cleaved, it will not be toxic. One skilled in the artwould predict that the molecule should, however, retain itsADP-ribosylating enzymatic activity and consequently, its adjuvantfunction.

FIG. 1 shows the sequence of the disulfide subtended region thatseparates the A₁ and A₂ pieces. Within this region is a single Arginineresidue which is believed to be the site of cleavage necessary toactivate the toxic properties of the molecule. This region was changedby site-directed mutagenesis is such a way as to render the moleculeinsensitive to proteolytic digestion and, consequently, nontoxic.

Site-directed mutagenesis is accomplished by hybridizing to singlestranded DNA a synthetic oligonucleotide which is complementary to thesingle stranded template except for a region of mismatch near thencenter. It is this region that contains the desired nucleotide change orchanges. Following hybridization with the single stranded target DNA,the oligonucleotide is extended with DNA polymerase to create a doublestranded structure. The nick is then sealed with DNA ligase and theduplex structure is transformed into an E. coli host. The theoreticalyield of mutants using this procedure is 50% due. to thesemi-conservative mode of DNA replication. In practice, the yield ismuch lower. There are, however, a number of methods available to improveyield and to select for oligonucleotide directed mutants. The systememployed utilized a second mutagenic oligonucleotide to create alteredrestriction sites in a double mutation strategy.

The next step was to substitute another amino acid for Arg (i.e.,GGA=Gly replaces AGA=Arg), thus preserving the reading frame whileeliminating the proteolytic site. mLT was then purified by agaroseaffinity chromatography from one mutant (pBD95) which had been confirmedby sequencing. Alternate methods of purification will be apparent tothose skilled in the art. This mutant LT, designated LT(_(R192G)) wasthen examined by SDS-polyacrylamide gel electrophoresis for modificationof the trypsin sensitive bond. Samples were examined with and withoutexposure to trypsin and compared with native (unmodified) LT. mLT doesnot dissociate into A₁ and A₂ when incubated with trypsin, therebyindicating that sensitivity to protease has been removed.

6.2 Effect of mLT on Y-1 Adrenal Cells

It would be predicted by one skilled in the art that mLT would not beactive in the Y-1 adrenal cell assay. This prediction would be basedupon previous findings [Clements and Finkelstein, 1979, Infect. Immun.24:760-769] that un-nicked LT was more than 1,000 fold less active inthis assay system than was CT and that trypsin treatment activated LT tothe same level of biological activity as CT in this assay. It waspresumed that the residual activity of LT observed in this assay in theabsence of trypsin activation was a function of some residual proteaseactivity which could not be accounted for. For instance, trypsin is usedin the process of subculturing Y-1 adrenal cells. It was thereforeassumed that LT that could not be nicked would be completely inactive inthe Y-1 adrenal cell assay. Results are shown in Table I.

TABLE I Toxin Trypsin Activated Specific Activity* Cholera Toxin − 15 LT− 60 LT + 15 LT_((R192G)) − 48,800 LT_((R192G)) + 48,800 *Minimum dose(picograms per well) required to produce (>50%) cell rounding.

Table I demonstrates the unexpected finding that mLT retained a basallevel of activity in the Y-1 adrenal cell assay even though it could notbe proteolytically processed. As shown in Table I, CT and native LTtreated with trypsin have the same level of activity (15 pg) on Y-1adrenal cells. By contrast, mLT (48,000 pg) was >1,000 fold less activethan CT or native LT and could not be activated by trypsin. The residualbasal activity undoubtedly reflects a different and here-to-fore unknownpathway of adrenal cell activation than that requiring separation of theA₁—A₂ linkage.

6.3 ADP-Ribosylating Enzymatic Activity of mLT

Because the mutation replacing Arg₁₉₂ with Gly₁₉₂ does not alter theenzymatic site of the A₁ moiety, one skilled in the art would predictthat mLT would retain its ADP-ribosylating enzymatic activity. Toexamine this property, the NAD-Agmatine ADP-ribosyltransferase Assay wasemployed [Moss et al., 1993, J. Biol. Chem. 268:6383-6387]. As shown inFIG. 2, CT produces a dose-dependent increase in the levels ofADP-ribosylagmatine, a function of the ADP-ribosyltransferase activityof this molecule.

TABLE II ADP-Ribosyltransferase Activity of CT, native LT, andLT_((R192G)) Experiment 1 2 3 4 Mean ± SEM No Toxin ND 9.12 5.63 14.179.64 ± 2.48 1 μgCT ND 17.81 17.60 25.75 20.39 ± 2.68  10 μgCT ND 107.32111.28 104.04 107.55 ± 2.09  100 μgCT 351.55 361.73 308.09 ND 340.46 ±16.45  100 μgLT 17.32 14.48 13.86 ND 15.22 ± 1.07  100 μgLT 164.10189.89 152.96 ND 168.98 ± 10.94  + Trypsin 100 μg 14.58 12.34 9.30 ND12.07 ± 1.53  LT_((R192G)) 100 μg 14.73 8.90 10.47 ND 11.37 ± 1.74 LT_((R192G)) + Trypsin ND = Not Done data expressed in fMoles min⁻¹

Table II demonstrates in tabular form the unexpected finding that mLTlacked any detectable ADP-ribosylating enzymatic activity, with orwithout trypsin activation, even though the enzymatic site had not beenaltered and there was a demonstratable basal level of activity in theY-1 adrenal cell assay.

6.4 Enterotoxiv Activity of mLT

Because of the unexpected finding that mLT lacks any detectableADP-ribosylating enzymatic activity, with or without trypsin activation,even though the enzymatic site has not been altered and the additionalfinding that there is a basal level of activity in the Y-1 adrenal cellassay, it was unclear whether mLT would retain any of its enterotoxicproperties. An ideal adjuvant formulation of mLT would retain itsability to act as an immunological adjuvant but would lack the real orpotential side-effects, such as diarrhea, associated with the use of LTor CT. FIG. 3 demonstrates that mLT does not induce net fluid secretionin the patent mouse model, even at a dose of 125 μg. This dose is morethan five times the adjuvant effective dose for LT in this model.Importantly, the potential toxicity of native LT can be seen at thislevel.

6.5 Adjuvant Activity of mLT

One skilled in the art would predict that since mLT possessed nodemonstrable ADP-ribosyltransferase activity and is not enterotoxic, itwould lack adjuvant activity. This prediction would be based upon thereport by Lycke et al. [Lycke et al., 1992, Eur. J. Immunol.22:2277-2281] where it is made clear that alterations that affect theADP-ribosylating enzymatic activity of the toxin and alter the abilityto increase intracellular levels of cAMP also prevent the molecule fromfunctioning as an adjuvant. As demonstrated above, mLT has noADP-ribosylating enzymatic activity and only some undefined basalactivity in Y-1 adrenal cells, and induces no net fluid secretion in thepatent mouse model.

In order to examine the adjuvant activity of mLT the followingexperiment was performed. Three groups of BALB/c mice were immunized.Animals were inoculated intragastrically with a blunt tipped feedingneedle (Popper & Sons, Inc., New Hyde Park, N.Y.). On day 0, each groupwas immunized orally as follows: Group A received 0.5 ml of PBScontaining 5 mg of OVA, Group B received 0.5 ml of PBS containing 5 mgof OVA and 25 μg of native LT, and Group C received 0.5 ml of PBScontaining 5 mg of OVA and 25 μg of mLT. Each regimen was administeredagain on days 7 and 14. On day 21, all animals were boosted i.p. with 1μg of OVA in 20% Maalox. One week after the i.p. inoculation animalswere sacrificed and assayed for serum IgG and mucosal IgA antibodiesdirected against OVA and LT by ELISA.

Reagents and antisera for the ELISA were obtained from Sigma ChemicalCo. Samples for ELISA were serially diluted in phosphate buffered saline(pH 7.2)-0.05% TWEEN™ (polyoxyethylenesorbitan monolaurate) 20(PBS-TWEEN™ (polyoxyethylenesorbitan monolaurate)). For anti-LTdeterminations, microtiter plates were precoated with 1.5 μg per well ofmixed gangliosides (Type III), then with 1 μg per well of purified LT.Anti-OVA was determined on microtiter plates precoated with 10 μg perwell of OVA. Serum anti-LT and anti-OVA were determined with rabbitantiserum against mouse IgG conjugated to alkaline phosphatase. Mucosalanti-LT and anti-OVA IgA were assayed with goat antiserum against mouseIgA (alpha-chain specific) followed by rabbit antiserum against goat IgGconjugated to alkaline phosphatase. Reactions were stopped with 3N NaOH.Values for IgG and IgA were determined from a standard curve withpurified mouse myeloma proteins (MOPC 315, gA(IgA12); MOPC 21, gG1:Litton Bionetics, Inc., Charleston, S.C.).

6.5.1 Serum IgG Anti-OVA

As shown in the FIG. 4A, animals primed orally with OVA and LT developeda significantly higher serum IgG anti-OVA response following subsequentparenteral immunization with OVA (4,058 μg/ml) than those primed withOVA alone and subsequently immunized parenterally with OVA (Nodetectable anti-OVA response) (Student t-test p=0.031). Significantly,animals primed orally with OVA and mLT also developed a significantlyhigher serum IgG anti-OVA response following subsequent parenteralimmunization with OVA (1,338 μg/ml) than those primed with OVA alone andsubsequently immunized parenterally with OVA (No detectable anti-OVAresponse) (Student t-test p=0.0007).

6.5.2 Mucosal sIgA Anti-OVA

As shown in the FIG. 4B, similar results were obtained when anti-OVA IgAresponses were compared within these same groups of animals. Animalsprimed orally with OVA and LT developed a significantly higher mucosalIgA anti-OVA response following subsequent parenteral immunization withOVA (869 ng/ml) than those primed with OVA alone and subsequentlyimmunized parenterally with OVA (No detectable anti-OVA response)(Student t-test p=0.0131). As above, animals primed orally with OVA andmLT also developed a significantly higher mucosal IgA anti-OVA responsefollowing subsequent parenteral immunization with OVA (230 ng/ml) thanthose primed with OVA alone and subsequently immunized parenterally withOVA (No detectable anti-OVA response) (Student t-test p=0.0189).

6.5.3 Serum IgG Anti-LT

The ability of LT and mLT to elicit an anti-LT antibody response inthese same animals was also examined. This was important in that itwould provide an indication of whether the mutant LT was able to preventinduction of tolerance to itself in addition to functioning as anadjuvant for other proteins. As shown in FIG. 5A, animals primed orallywith OVA and LT developed a significantly higher serum IgG anti-LTresponse following subsequent parenteral immunization with OVA (342μg/ml) than those primed with OVA alone and subsequently immunizedparenterally with OVA (No detectable anti-LT response) (Student t-testp=0.0005). Animals primed orally with OVA and mLT also developed asignificantly higher serum IgG anti-LT response following subsequentparenteral immunization with OVA (552 μg/ml) than those primed with OVAalone and subsequently immunized parenterally with OVA (No detectableanti-LT response) (Student t-test p=0.0026).

6.5.4 Mucosal sIgA Anti-LT

As shown in the FIG. 5B, similar results were obtained when anti-LT IgAresponses were compared within these same groups of animals. Animalsprimed orally with OVA and LT developed a significantly higher mucosalIgA anti-LT response following subsequent parenteral immunization withOVA (4,328 ng/ml) than those primed with OVA alone and subsequentlyimmunized parenterally with OVA (No detectable anti-LT response)(Student t-test p=0.0047). As above, animals primed orally with OVA andmLT also developed a significantly higher mucosal IgA anti-LT responsefollowing subsequent parenteral immunization with OVA (1,463 ng/ml) thanthose primed with OVA alone and subsequently immunized parenterally withOVA (No detectable anti-LT response) (Student t-test p=0.0323).

7. DEPOSIT OF MICROORGANISMS

The following plasmid was deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209,on Aug. 18, 1994, and has been assigned the indicated accession number:

Plasmid Accession Number pBD95 ATCC 69683

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed since these embodiments areintended as illustration of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

It is also to be understood that all base pair and amino acid residuenumbers and sizes given for nucleotides and peptides are approximate andare used for purposes of description.

A number of references are cited herein, the entire disclosures of whichare incorporated herein, in their entirety, by reference.

5 45 base pairs nucleic acid double unknown DNA 1 GGTTGTGGAA ATTCATCAAGAACAATTACA GGTGATACTT GTAAT 45 15 amino acids amino acid linear protein2 Gly Cys Gly Asn Ser Ser Arg Thr Ile Thr Gly Asp Thr Cys Asn 1 5 10 1518 base pairs nucleic acid double unknown DNA 3 TCATCAGGAA CAATTACA 1845 base pairs nucleic acid double unknown DNA 4 GGTTGTGGAA ATTCATCAGGAACAATTACA GGTGATACTT GTAAT 45 15 amino acids amino acid linear protein5 Gly Cys Gly Asn Ser Ser Gly Thr Ile Thr Gly Asp Thr Cys Asn 1 5 10 15

What is claimed is:
 1. A vaccine preparation comprising an antigen incombination with a composition comprising a mutant E. coli heat-labileenterotoxin holotoxin, in which arginine at amino acid position 192 isreplaced with glycine, which holotoxin is substantially less toxic thannative E. coli heat-labile enterotoxin holotoxin as measured in the Y-1adrenal cell assay and which has immunologic adjuvant activity but lacksADP-ribosylating enzymatic activity as measured by the NAD-AgmatineADP-ribosyltransferase assay.
 2. The vaccine preparation of claim 1, inwhich the antigen is an antigen of a bacterium selected from the groupconsisting of Streptococcus pyogenes, Streptococcus pneumoniae,Neisseria gonorrheae, Neisseria meningitidis, Corynebacteriumdiphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridiumtetani, Hemophilus influenzae, Klebsiella pheumoniae, Klebsiellaozaenae, Klebsiella rhinoscleromatis, Staphylococcus aureus, Vibriocholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter(Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiellatarda, Yersinia enterocolitica, Yersinia pestis, Yersiniapseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigellasonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue,Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi,Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Francisellatularensis, Brucella abortus, Brucella suis, Brucella melitensis,Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugamushi, andChlamydia spp.
 3. The vaccine preparation of claim 1, in which theantigen is an antigen of a fungus selected from the group consisting ofCoccidioides immitis, Aspergillus fumigatus, Candida albicans,Blastomyces dermatitidis, Cryptococcus neoformans, and Histoplasmacapsulatum.
 4. The vaccine preparation of claim 1, in which the antigenis an antigen of a protozoan selected from the group consisting ofEntomoeba histolytica, Trichomonas tenas, Trichomonas hominis,Trichomonas vaginalis, Trypanosoma gambiense, Trypanosoma rhodesiense,Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi,Leishania donovani, Leishamania tropica, Leishmania braziliensis,Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, andPlasmodium malaria.
 5. The vaccine preparation of claim 1, in which theantigen is an antigen of a Helminth selected from the group consistingof Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides,Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum,Schistosoma mansoni, Schistosoma haematobium, and hookworm.
 6. Thevaccine preparationof claim 1, in which the antigen is an antigen of avirus selected from the group consisting of Poxviridae, Herpesviridae,Herpes Simplex virus 1, Herpes Simples virus 2, Adenoviridae,Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae,retroviridae, influenza viruses, parainfluenza viruses, mumps, measles,respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae,Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus,Hepatitis E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae,Coronaviridae, and Rotaviridae.
 7. A composition comprising (a) avaccine selected from the group consisting of influenza vaccine,pertussis vaccine, diphtheria and tetanus toxoid combined with pertussisvaccine, hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine,hepatitis E vaccine, Japanese encephalitis vaccine, herpes vaccine,measles vaccine, rubella vaccine, mumps vaccine, mixed vaccine ofmeasles, mumps and rubella, papillomavirus vaccine, parvovirus vaccine,respiratory syncytial virus vaccine, Lyme disease vaccine, poliovaccine, malaria vaccine, varicella vaccine, gonorrhea vaccine,schistosomiasis vaccine, rota vaccine, Campylobacter vaccine, choleravaccine, enteropathogenic E. coli vaccine, enterotoxic E. coli vaccine,mycoplasma vaccine, pneumococcal vaccine, and meningococcal vaccine, and(b) a composition comprising a mutant E. coli heat-labile enterotoxinholotoxin, in which arginine at amino acid position 192 is replaced withglycine, which holotoxin is substantially less toxic than native E. coliheat-labile enterotoxin holotoxin as measured in the Y-1 adrenal cellassay and which has immunologic adjuvant activity but lacksADP-ribosylating enzymatic activity as measured by the NAD-AgmatineADP-ribosyltransferase assay.
 8. A kit useful in producing a protectiveimmune response in a host to a pathogen comprising two components: (a)an effective amount of a protective antigen of a bacterial, viral orfungal pathogen, and (b) an adjuvant effective amount of a mutant E.coli heat-labile enterotoxin holotoxin, in which arginine at amino acidposition 192 is replaced with glycine, and which has immunologicadjuvant activity but lacks ADP-ribosylating enzymatic activity asmeasured by the NAD-Agmatine ADP-ribosyltransferase assay, wherein bothsaid components are in an orally acceptable carrier and said componentsmay be administered either after having been mixed together orseparately.